Engineered viral vector reduces induction of inflammatory and immune responses

ABSTRACT

Modified viral genomes are able to reduce induction of inflammatory and immune anti-viral responses. This manifests itself in reduced NF-kB activity, increased viral transduction rates, and increased expression of transgenes. Viral genomes are modified by incorporating one or more oligonucleotide sequences which are able to bind to TLR9 but not induce activation of it. The oligonucleotide sequences may be synthetic, bacterial, human, or from any other source.

RELATED APPLICATION DATA

This application is continuation of PCT/US2017/036525 filed Jun. 8,2017, which claims priority to U.S. Provisional Application No.62/347,302 filed on Jun. 8, 2016, each of which is herein incorporatedby reference in its entirety for all purposes.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. HG008525awarded by National Institutes of Health. The government has certainrights in the invention.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the area of viral based therapies. Inparticular, it relates to recombinant viruses.

BACKGROUND OF THE INVENTION

Gene therapy has immense potential to prevent, treat, and cure multiplehuman diseases [1]. In 2012, Glybera™ (alipogene tiparvovec) became thefirst viral gene therapy to be approved for use in the western world[2]. Glybera™ utilizes adeno-associated virus (AAV) as a viral vector todeliver the human lipoprotein lipase (LPL) gene to muscle cells inpatients with LPL deficiency [3]. Many AAV clinical trials are currentlyunderway or being planned in the U.S. and the EU [4-6].

AAV is a small, non-enveloped virus that packages a single-strandedlinear DNA genome that is approximately 5 kb long, and has been adaptedfor use as a gene transfer vehicle [4]. The coding regions of AAV areflanked by inverted terminal repeats (ITRs), which act as the originsfor DNA replication and serve as the primary packaging signal [7, 8].Both positive and negative strands are packaged into virions equallywell and capable of infection [9-11]. In addition, a small deletion inone of the two ITRs allows packaging of self-complementary vectors, inwhich the genome self-anneals after viral uncoating. This results inmore efficient transduction of cells but reduces the coding capacity byhalf [12, 13]. While AAV is not associated with any human disease, >70%of humans are seropositive for one or most serotypes [14, 15]. Typicalroutes of administration for AAV vectors include intravenous,intramuscular, subretinal and intracranal injections. AAV gene therapyis most often used to deliver a wild-type gene to treat monogenicdiseases, and AAV vectors have been used to transduce cells in theliver, skeletal and cardiac muscle, retina and central nervous system[16-26]. In addition, there is now growing interest in using AAV todeliver CRISPR-Cas9 gene editing or to deliver broadly neutralizingantibodies against infectious diseases such as human immunodeficiencyvirus (HIV) and influenza A virus [27-32].

Despite several advances in gene therapy, a key concern is theinflammatory response elicited by the viral vector [33, 34]. This isbest illustrated by a highly publicized case in 1999 when JesseGelsinger died four days after gene therapy with adenovirus in aclinical trial due to excessive inflammation [35]. While AAV has beenshown to elicit much weaker inflammation in comparison to adenoviruses[36, 37], Glybera™ therapy still includes twelve weeks ofimmunosuppression, beginning three days before Glybera™ administration[38]. These immunosuppressive and anti-inflammatory drugs (cyclosporineA, mycophenolate mofetil, and methylprednisolone) compromise thepatient's immune system during treatment, and all patients stilldeveloped neutralizing antibodies against AAV capsid, precluding futurere-administration. Furthermore, many AAV gene therapy clinical trials donot utilize immunosuppression prophylactically and only administercorticosteroids upon signs of inflammation or tissue damage, which hasbeen associated with variable therapeutic efficacy [22, 23].Interestingly, in agreement with their ability to trigger inflammatoryand immune responses, AAV vectors have also been developed as vaccinevehicles against infectious diseases and cancer [39-41].

Inflammation has been implicated as a critical determinant of successfulAAV-mediated transgene expression. A study found that following AAVadministration in mice, artificially inducing systemic inflammation suchas the upregulation of tumor necrosis factor (TNF), led to a decline intransgene expression in the liver [42]. This indicates thatimmunological tolerance to AAV-encoded transgene can be broken givensufficient inflammatory responses. Another study characterizedinflammation in the murine liver following AAV infection and foundtransient increases in liver enzymes in the serum, and also liverpathology consistent with portal and lobular inflammation [43].Strikingly, the authors observed that the use of AAVrh.32.33 viruses,which induced higher liver enzymes than other tested AAV serotypes, alsoled to a decline in transgene expression to below detection levels,again suggesting that inflammatory and immune responses are associatedwith poor transgene expression.

In hemophilia B clinical trials, it has been observed that a subset ofpatients exhibited elevated liver enzymes, and this transienttransaminitis was accompanied by declining levels of transgene-encodedfactor IX [22, 23]. A tapering course of corticosteroid therapy was usedon a patient with transaminitis, which subsequently normalizedaminotransferase levels in the serum and rescued further decline offactor IX expression. Overall, these observations are compatible withimmune-mediated destruction of AAV-transduced hepatocytes anddemonstrate that inflammatory and immune responses triggered by AAVadministration are a safety concern and can hamper therapeutic efficacyin humans. Thus, it would be advantageous to develop viral vectors thatintrinsically evade eliciting inflammation. Furthermore, instead ofsystemic immunosuppression with drugs, it would be beneficial to avoidtriggering specific immune responses.

It has been previously shown that the DNA genome of AAV is sensed byToll-like receptor 9 (TLR9) during AAV's entry into the cell through theendocytic pathway [36, 44]. TLR9 is a pattern recognition receptor (PRR)found on endosomal membranes of immune cells such as B cells, monocytes,macrophages and plasmacytoid dendritic cells, and binds to unmethylatedCpG motifs found in the AAV genome [45, 46]. This leads to TLR9dimerization, which triggers a cascade of signal transduction thatactivates NF-kB (also known as p52-RelA complex) and induces type Iinterferons (IFNs). NF-kB in turn drives the transcriptionalupregulation of multiple proinflammatory cytokines such as TNF leadingto inflammation and immune cell recruitment, while secreted IFNs inducethe expression of numerous interferon-stimulated genes (ISGs) andestablish an antiviral state. Importantly, genetic ablation of TLR9 inmice abolishes induction of inflammatory cytokines upon AAV treatment inthe liver, and also reduces formation of antibodies and T cells againstAAV [36]. Thus, TLR9 plays a critical role in stimulating an earlyinflammatory and innate immune response during AAV infection, which alsocontributes to priming adaptive immunity. Finally, two other patternrecognition receptors, TLR2 and TLR4, have been implicated in triggeringresponses to AAV structural proteins [47, 48].

In the TLR9 field, a commonly used tool to block TLR9 activation in cellculture is short, single-stranded DNA oligonucleotides that bind TLR9but do not activate it [49, 50]. Several such sequences are known—somesynthetic and others derived from organisms—and they often bear nosequence homology [51-59]. Structural studies have revealed how aninhibitory oligonucleotide binds TLR9 tightly but does not trigger TLR9dimerization, which is required for TLR9 activation and downstreamsignaling [60]. In addition to binding TLR9 directly to antagonize itsactivation, other mechanisms to block TLR9 activation or TLR9-mediatedinflammation have been postulated or shown for other TLR9-inhibitoryoligonucleotides [reviewed in 49]. These include competing forreceptor-mediated endocytosis or phagocytosis, inhibition of TLR9trafficking or TLR9 processing into a functionally active product,inhibition of endosomal acidification or activity of key proteases inendosomes, or blocking signaling proteins downstream of TLR9. When theseinhibitory oligonucleotides are supplied in trans with TLR9 ligands(such as a DNA virus, or a CpG-containing oligonucleotide) in cellculture media, they are endocytosed and can bind to TLR9, preventing itsactivation by stimulatory ligands. Supplementation of inhibitoryoligonucleotides in trans is widely adopted in immunology experiments,but it is unknown in the field if incorporation of these sequences intoa viral genome allows it to evade eliciting inflammatory and immuneresponses.

While AAV has been shown to elicit much weaker inflammatory responses incomparison to adenoviruses, Glybera™ alipogene tiparvovec treatmentstill includes twelve weeks of immunosuppression, beginning three daysbefore Glybera™ alipogene tiparvovec administration. Theseimmunosuppressive drugs strongly hamper T cell activation and thereforecompromise the patient's immune system during treatment. It would beadvantageous to engineer viral vectors that evade and elicit diminishedor no inflammatory response upon administration. Furthermore, it wouldbe beneficial if the immune suppression was not systemic, and if it wastransient. Preventing inflammatory and immune responses could alsoimprove transgene expression and may allow the re-administration of theviral vector for future purposes.

There is a continuing need in the art to improve the efficacy of viralvectors for therapy and for in vivo production of biological products.

SUMMARY OF THE INVENTION

According to one aspect of the invention a nucleic acid molecule isprovided. It comprises a viral genome covalently linked to an inhibitorynucleic acid sequence which binds to TLR9 but does not trigger TLR9activation.

According to another aspect a recombinant virus is provided for deliveryof a desired function to a mammalian cell. The recombinant viruscomprises a viral genome covalently linked to an inhibitory nucleic acidsequence which binds to TLR9 but does not trigger TLR9 activation.

Another embodiment is an aspect of treating a mammal. The methodcomprises administering a recombinant virus to a mammal in need thereof.The recombinant virus comprises a viral genome covalently linked to aninhibitory nucleic acid sequence which binds to TLR9 but does nottrigger TLR9 activation.

Still another aspect is a method of making a viral genome of arecombinant virus. An inhibitory nucleic acid sequence is inserted intoa viral genome. The inhibitory nucleic acid sequence binds to TLR9 butdoes not trigger TLR9 activation.

According to one aspect a nucleic acid molecule is provided. Itcomprises an inverted terminal repeat (ITR) and a nucleic acid sequencewhich inhibits TLR9-mediated inflammation.

Another aspect of the invention is a nucleic acid molecule. The moleculecomprises a viral genome covalently linked to an inhibitory nucleic acidsequence which inhibits TLR9-mediated inflammation.

Yet another aspect of the invention is a recombinant virus for deliveryof a desired function to a mammalian cell. The recombinant viruscomprises a viral genome comprising an inhibitory nucleic acid sequencewhich inhibits TLR9-mediated inflammation.

Still another aspect of the invention is a method of making a viralgenome of a recombinant virus. A nucleic acid sequence is inserted intoa viral genome. The nucleic acid sequence inhibits TLR9-mediatedinflammation.

According to another aspect of the invention a nucleic acid vector isprovided. The vector comprises at least one nucleic acid sequence. Thenucleic acid sequence is capable of inhibiting TLR9-mediatedinflammation.

Another aspect of the invention is a method of reducing immunogenicityof a modified virus having a genome. The method comprises inserting anucleic acid sequence into the genome. The nucleic acid sequenceinhibits TLR9-mediated inflammation. The modified virus causes a reducedinflammatory response in a host as compared to a virus that does notcontain the inhibitory sequence.

Still another aspect of the invention is a method of increasingexpression in a host cell of a virally introduced transgene. The methodcomprises introducing into a host a modified virus having a genome. Thegenome comprises a nucleic acid sequence. The nucleic acid sequenceinhibits TLR9-mediated inflammation. The modified virus results inhigher transgene expression in a host cell as compared to a virus thatdoes not contain the inhibitory sequence.

Yet another aspect of the invention is a composition comprising a viralcapsid encapsidating a nucleic acid sequence that inhibits TLR9-mediatedinflammation.

These and other aspects and embodiments which will be apparent to thoseof skill in the art upon reading the specification provide the art withtools for better treating mammals with viral vectors and virions and forbetter using viral vectors and virions for producing products oftransgenes in host cells, host tissues, and host animals.

Any and all of the above described aspects may be combined with any ofthe following features.

The viral genome may be adeno-associated virus (AAV) genome.

The viral genome may be selected from the group consisting ofadenovirus, herpes simplex virus, varicella, variola virus, hepatitis B,cytomegalovirus, JC polyomavirus, BK polyomavirus, monkeypox virus,Herpes Zoster, Epstein-Barr virus, human herpes virus 7, Kaposi'ssarcoma-associated herpesvirus, and human parvovirus B19.

The viral genome may be single stranded.

The viral genome may be packaged in a virion.

The viral genome may comprise a gene which may be expressible in a humancell.

The viral genome may be a cytotoxic virus for lysing target tumor cells.

The inhibitory nucleic acid sequence may comprise c41 oligonucleotidesequence TGGCGCGCACCCACGGCCTG (SEQ ID NO: 1).

The inhibitory nucleic acid sequence may comprise a plurality of copiesof c41 sequence (SEQ ID NO: 1).

The inhibitory nucleic acid sequence may comprise two copies of c41sequence (SEQ ID NO: 1) separated by a linker sequence.

The linker sequence is AAAAA (SEQ ID NO: 8).

The inhibitory nucleic acid sequence may be selected from the groupconsisting of:

ODN 2088: (SEQ ID NO: 2) TCC TGG CGG GGA AGT; ODN 4084-F: (SEQ ID NO: 3)CCTGGATGGGAA; ODN INH-1: (SEQ ID NO: 4) CCTGGATGGGAATTCCCATCCAGG;ODN INH-18: (SEQ ID NO: 5) CCT GGA TGG GAA CTT ACC GCT GCA; ODN TTAGGG:(SEQ ID NO: 6) TT AGG GTT AGG GTT AGG GTT AGG G; and G-ODN:(SEQ ID NO: 7) CTC CTA TTG GGG GTT TCC TAT.

The inhibitory nucleic acid sequence may be a bacterial sequence.

The viral genome, recombinant virus, vector, or nucleic acid sequencemay comprise a non-human gene.

The inhibitory nucleic acid sequence may be inserted downstream of or ina 3′ untranslated region of the viral genome.

The viral genome may be covalently linked to the inhibitory nucleic acidsequence by a phosphodiester bond.

The viral genome, recombinant virus, vector, or nucleic acid sequencemay comprise a detectable marker.

The detectable marker may be inducible.

The inhibitory nucleic acid sequence may comprise a human telomeresequence shown in SEQ ID NO: 9.

The viral genome may be self-complementary.

The viral genome may be covalently linked to a plurality of inhibitorynucleic acid sequences.

The plurality of inhibitory nucleic acid sequences may comprise aninhibitory sequence and its reverse complement.

The inhibitory nucleic acid sequence may comprise three copies of c41sequence (SEQ ID NO: 1), each copy separated by a linker sequence.

The inhibitory nucleic acid sequence may be selected from the groupconsisting of:

ODN 2114: (SEQ ID NO: 16) TCCTGGAGGGGAAGT; ODN 4024: (SEQ ID NO: 17)TCCTGGATGGGAAGT; ODN INH-4: (SEQ ID NO: 18) TTCCCATCCAGGCCTGGATGGGAA;ODN INH-13: (SEQ ID NO: 19) CTTACCGCTGCACCTGGATGGGAA; ODN Poly-G:(SEQ ID NO: 20) GGGGGGGGGGGGGGGGGGGG; ODN GpG: (SEQ ID NO: 21)TGACTGTGAAGGTTAGAGATGA; ODN IRS-869: (SEQ ID NO: 22) TCCTGGAGGGGTTGT;ODN IRS-954: (SEQ ID NO: 23) TGCTCCTGGAGGGGTTGT; and ODN 21158:(SEQ ID NO: 24) CCTGGCGGGG.

The inhibitory nucleic acid sequence may be ODN TTAGGG (SEQ ID NO: 6).

The inhibitory sequence may be covalently linked to a linker.

The inhibitory sequence may be upstream of the linker.

The inhibitory nucleic acid sequence may comprise a plurality of copiesof ODN TTAGGG (SEQ ID NO: 6).

The plurality of copies of ODN TTAGGG (SEQ ID NO: 6) may each beseparated by a linker.

The inhibitory nucleic acid sequence may comprise at least 2, at least3, at least 4, or at least 5 copies of ODN TTAGGG (SEQ ID NO: 6), eachcopy separated by a linker.

The inhibitory nucleic acid sequence may be a human sequence.

The viral genome, recombinant virus, vector, or nucleic acid sequencemay comprise a non-human nucleic acid sequence.

The viral genome, recombinant virus, vector, or nucleic acid sequencemay comprise a human gene.

The viral genome, recombinant virus, vector, or nucleic acid sequencemay comprise a human nucleic acid sequence.

The inhibitory nucleic acid sequence may be inserted in the 5′untranslated region of the viral genome.

The inhibitory nucleic acid sequence may be inserted upstream of apromoter of the viral genome.

The viral genome, recombinant virus, vector, or nucleic acid sequencemay comprise an inducible promoter.

The inhibitory nucleic acid sequence may comprise two repeated monomersof SEQ ID NO: 1.

The inhibitory nucleic acid sequence may comprise three repeatedmonomers of SEQ ID NO: 1.

The inhibitory nucleic acid sequence may comprise SEQ ID NO: 6 or SEQ IDNO: 9.

The inhibitory nucleic acid sequence may comprise three repeatedmonomers of SEQ ID NO: 6 or SEQ ID NO: 9.

The inhibitory nucleic acid sequence may comprise five repeated monomersof SEQ ID NO: 6 or SEQ ID NO: 9.

The step of administering may be repeated.

The viral genome may be packaged in virions.

The step of inserting may utilize a DNA ligase.

The viral genome may be single stranded when in virions.

The viral genome of the recombinant virus may comprise a gene fordelivery to and expression in a human cell.

The viral genome, recombinant virus, vector, or nucleic acid sequencemay comprise a linker separating each of the nucleic acid sequences.

The viral genome, recombinant virus, vector, or nucleic acid sequencemay comprise at least 2, at least 3, at least 4, or at least 5 copies ofthe nucleic acid sequence.

The inhibitory nucleic acid may be covalently linked to a gene.

The inhibitory nucleic acid sequence may be 95% identical to c41oligonucleotide sequence TGGCGCGCACCCACGGCCTG (SEQ ID NO: 1).

The inhibitory nucleic acid sequence may be 95% identical to SEQ ID NO:9.

The inhibitory nucleic acid sequence may be 95% identical to a sequenceselected from the group consisting of:

ODN 2088: (SEQ ID NO: 2) TCC TGG CGG GGA AGT; ODN 4084-F: (SEQ ID NO: 3)CCTGGATGGGAA; ODN INH-1: (SEQ ID NO: 4) CCTGGATGGGAATTCCCATCCAGG;ODN INH-18: (SEQ ID NO: 5) CCT GGA TGG GAA CTT ACC GCT GCA; ODN TTAGGG:(SEQ ID NO: 6) TT AGG GTT AGG GTT AGG GTT AGG G; and G-ODN:(SEQ ID NO: 7) CTC CTA TTG GGG GTT TCC TAT.

The inhibitory nucleic acid sequence may be 95% identical to a sequenceselected from the group consisting of:

ODN 2114: (SEQ ID NO: 16) TCCTGGAGGGGAAGT; ODN 4024: (SEQ ID NO: 17)TCCTGGATGGGAAGT; ODN INH-4: (SEQ ID NO: 18) TTCCCATCCAGGCCTGGATGGGAA;ODN INH-13: (SEQ ID NO: 19) CTTACCGCTGCACCTGGATGGGAA; ODN Poly-G:(SEQ ID NO: 20) GGGGGGGGGGGGGGGGGGGG; ODN GpG: (SEQ ID NO: 21)TGACTGTGAAGGTTAGAGATGA; ODN IRS-869: (SEQ ID NO: 22) TCCTGGAGGGGTTGT;ODN IRS-954: (SEQ ID NO: 23) TGCTCCTGGAGGGGTTGT; and ODN 21158:(SEQ ID NO: 24) CCTGGCGGGG.

The inhibitory nucleic acid sequence may comprise a plurality of copiesof SEQ ID NO: 6 and/or SEQ ID NO: 9

The inhibitory nucleic acid sequence may comprise two copies of SEQ IDNO: 6 and/or SEQ ID NO: 9 separated by a linker sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic drawing of viral gene therapy and inflammatory andimmune responses.

FIG. 2. Schematic drawing showing TLR9 sensing AAV DNA during viralentry and inflammatory and immune responses. From Rogers et al., 2011,Frontiers in Microbiology, “Innate Immune Responses to AAV Vectors,”vol. 2, article 194.

FIGS. 3A-3B. FIG. 3A shows nucleotide sequence of c41 (SEQ ID NO: 1), asingle-stranded oligonucleotide. FIG. 3B shows organization of AAV-eGFPand AAV-eGFP-c41 genomes. ITR, inverted terminal repeat; LPA, latepolyadenylation signal. Transgene of interest depicted in this case iseGFP. The insertion is of c41 sequence. Spacer depicted in FIG. 3B isshown in SEQ ID NO: 8.

FIG. 4 shows NF-kB activity in HEK293 TLR9 cells mock-infected orinfected with AAV virus (DJ capsid, self-complementary AAV genome,encoding eGFP). **, p<0.005; n.s., not significant. This experiment useda crude viral preparation.

FIG. 5. Flow cytometry histograms showing GFP expression in HEK293 TLR9cells mock-infected or infected with AAV virus with or without c41. Thisexperiment used a crude viral preparation.

FIGS. 6A-6B. FIG. 6A shows nucleotide sequence of “telomere” (SEQ ID NO:9), a single-stranded oligonucleotide containing the (TTAGGG)₄ (SEQ IDNO: 6) motif from mammalian telomeres. FIG. 6B shows organization ofAAV-eGFP-telomere genome. The insertion is of “telomere” sequence.Spacers shown in FIG. 6B are SEQ ID NO: 8.

FIGS. 7A-7B. FIG. 7A shows the percentage of transduced cells (GFP+) 2days after infection of a B cell line with similar amounts of indicatedAAV viruses, analyzed by flow cytometry. FIG. 7B shows TNF production inthe supernatant of primary human CD14+ monocytes 18 hours afterinfection, as assayed by ELISA.

FIG. 8A-8B. Engineering a self-complementary AAV vector. (FIG. 8A) DNAsequences of “c41” and “telomere”. (FIG. 8B) Genome organization of anAAV vector (scAAV-eGFP) and modified vectors. LpA: polyA signal.

FIG. 9A-9C Inflammatory response to various AAV vectors in human immunecells in vitro. (FIG. 9A) Primary human macrophages were infected withAAV2 viruses (MOI: 10⁵ vg/cell) and supernatants were collected 18 hlater and analyzed by ELISA for TNF levels. Five uM ODN 2006, aCpG-containing oligonucleotide, served as a positive control. (FIG. 9B,FIG. 9C) Primary human CD14+ monocytes from two different donors wereinfected similar to (A) and analyzed for TNF levels. Data shown aremean±s.d. of n=3 technical replicates. *P<0.05 (unpaired t-test)compared to scAAV-eGFP.

FIG. 10A-10E. Further characterization of AAV vectors. (FIG. 10A) DNAsequence of “control”. (FIG. 10B) Genome organization of modifiedvectors. (FIG. 10C) Primary human macrophages were infected with AAV2viruses similar to (FIG. 9A) and analyzed by ELISA for TNF levels. (FIG.10D) Primary human monocytes were infected similar to (FIG. 9B, FIG. 9C)and analyzed by ELISA for TNF levels. Data shown (FIG. 10C, FIG. 10D)are mean±s.d. of n=3 technical replicates. *P<0.05 (unpaired t-test)compared to scAAV-eGFP. (FIG. 10E) Adult C57BL/6 mice were infected withindicated AAV2 viruses similar to (FIG. 12A, FIG. 12B and FIG. 12C) anda piece of the liver was analyzed for indicated gene expression byqRT-PCR. Data shown are mean±s.d. of n=5 mice per condition except n=3mice for scAAV-eGFP-3×control. *P<0.05 (unpaired t-test) compared tosaline condition. N.s.: not significant (P>0.05).

FIG. 11A-11B. (FIG. 11A) Primary human macrophages using a different lotof both AAV2 viruses were infected similar to (FIG. 9A) and analyzed forTNF levels. (FIG. 11B) HeLa cells were infected with AAV2 viruses atindicated MOIs and cells were harvested 48 h later and analyzed by flowcytometry for GFP expression. The percentage of GFP positive cells areshown Data shown are mean±s.d. of n=3 technical replicates. *P<0.05(unpaired t-test) compared to scAAV-eGFP.

FIG. 12A-12C Inflammatory response to intravenous administration ofvarious AAV vectors in adult mice in vivo. (FIG. 12A, FIG. 12B and FIG.12C) Adult C57BL/6 mice were infected with indicated AAV2 viruses (10¹¹vg per mouse) by tail vein injections. 2 h later, the animals wereeuthanized and a piece of the liver was analyzed for indicated geneexpression by qRT-PCR. Saline injection was set to 1-fold expression foreach gene. Data shown are mean±s.d. of n=3 mice per condition (FIG. 12A)or n=4 mice per condition (FIG. 12B and FIG. 12C). *P<0.05 (unpairedt-test) compared to saline condition. N.s.: not significant (P>0.05).

FIG. 13. Genome organization of a single-stranded AAV vector(ssAAV-eGFP) and ssAAV-eGFP-5× telomere.

FIG. 14A-14C. Inflammatory and immune response following subretinaladministration of various AAV vectors in neonatal mice in vivo. (FIG.14A, FIG. 14B and FIG. 14C) Neonatal CD1 mice (P1) received indicatedAAV8 viruses (1.8×10⁸ vg per mouse eye) by subretinal injections. AtP21, the animals were euthanized and the eyecup was dissected out. Theretina and the rest of the eyecup were analyzed for indicated geneexpression by qRT-PCR. Saline injection was set to 1 fold expression foreach gene. Each triangle represents an animal. Data shown are n=3 mice(saline) and n=5 mice (each virus) and mean values are indicated.

FIG. 15A-15C. Analysis of immune cell markers in the retina followingsubretinal administration of various AAV vectors in neonatal mice invivo. (FIG. 15A, FIG. 15B and FIG. 15C) Similar to (FIG. 14A-14C), theretina was analyzed for indicated gene expression by qRT-PCR. Aif1(Iba1) is known to be expressed in microglia, while Cd4 and Cd8a aremarkers of helper and cytolytic T cells respectively. Saline injectionwas set to 1 fold expression for each gene. Each triangle represents ananimal. Data shown are n=3 mice (saline) and n=5 mice (each virus) andmean values are indicated.

FIG. 16. GFP expression in the eye. Neonatal CD1 mice (P1) receivedindicated AAV8 viruses (1.8×10⁸ vg per mouse eye) by subretinalinjections. At P30, the animals were euthanized and GFP expression wasvisualized in flat-mounted eye cups. Data shown are n=2 mice percondition.

FIGS. 17A-17B. FIG. 17A shows nucleotide sequence of c41 (SEQ ID NO: 1),a single-stranded oligonucleotide. FIG. 17B shows organization ofAAV-eGFP and AAV-eGFP-c41 genomes. ITR, inverted terminal repeat; LPA,late polyadenylation signal. Transgene of interest depicted in this caseis eGFP. The insertion is of c41 sequence. Spacer depicted in FIG. 17Bis shown in SEQ ID NO: 8.

FIGS. 18A-18B. FIG. 18A shows nucleotide sequence of “telomere” (SEQ IDNO: 9), a single-stranded oligonucleotide containing the (TTAGGG)₄ (SEQID NO: 6) motif from mammalian telomeres. FIG. 18B shows organization ofAAV-eGFP-telomere genome. The insertion is of “telomere” sequence.Spacers shown in FIG. 18B are SEQ ID NO: 8.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have developed viral vectors and virions that harbor theirown protection against host immune and inflammatory systems. Thesevectors and virions carry short nucleic acid sequences which inhibit theactivation of toll-like receptor 9 (TLR9), a host protein whichactivates inflammatory and immune responses in mammalian cells.

A short nucleotide sequence for inhibition of TLR9 may be of any origin.It can be bacterial, human, synthetic, or from other sources. Oneparticular sequence is the 20 nucleotide long “c41”[TGGCGCGCACCCACGGCCTG (SEQ ID NO: 1)] from Pseudomonas aeruginosa.Another particular sequence is from human telomeres and comprises(TTAGGG)₄ (SEQ ID NO:6). Other inhibitory sequences are shown in SEQ IDNO: 2-5, 7, 9, and 16-24. Inhibitory sequences may also be used whichshare at least 80% homology/identity with these sequences. Inhibitorysequences may also be used which share at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, and/or at least 99%homology/identity with these sequences. Multiple copies of theinhibitory sequence can be used, either in tandem arrays or separated inthe viral vector by spacer or linker sequences or other portions of theviral genome. In some embodiments one, two, three, four, five, six,seven, eight, nine, ten, fifteen, or twenty copies are used. In someembodiments one or more copies of the inhibitory sequence are on theplus strand and some on the minus strand of the virus genome.

The inhibitory oligonucleotide sequences are introduced into host cellsas part of the viral genome or virion, rather than as a separate agent.This renders the effect of the oligonucleotide sequences local ratherthan systemic. Moreover, the immune evasion is transient as it occursduring AAV or other virus entry, unlike immune suppression with drugswhich can last for weeks. Additionally, it ensures that the beneficialantagonist activity is where it needs to be—with the virus or viralgenome. If the virus or viral genome is not replicated in the host cell,then the effect of the oligonucleotide will be transient. If the virusor viral genome is replicated, the effect will be coextensive with thereplication.

An inhibitory nucleic acid sequence may be inserted into a viral genomeusing any means of recombinant DNA engineering. This may involve invitro or in vivo recombination. In vitro recombination may beaccomplished using a DNA ligase or other nucleic acid joining enzyme,for example. In vivo recombination may be accomplished byco-transforming a host cell with separate donor molecules that sharehomology by which they will recombine using host cell machinery.Alternatively, a single donor molecule may recombine in vivo with a hostcell sequence. Combinations of these approaches may also be used.Typically the insertion will involve a standard linkage of onedeoxyribonucleotide to another (a phosphodiester bond). However, theremay be circumstances in which non-standard linkages will be used betweenthe inhibitory nucleic acid sequence and the rest of the viral genome.Optionally, the inhibitory nucleic acid sequence is located in anuntranslated region of the viral genome.

The genome may optionally contain a therapeutic gene and/or a markergene. Typically this gene will be a non-viral gene, or a gene that isnot naturally present in the viral genome. The gene may be expressiblein a mammalian host cell or animal. Expression may be under the controlof a viral promoter or a promoter that is introduced with the gene.Expression may be inducible, repressible, condition-responsive, orconstitutive, as examples. A therapeutic gene is one which encodes anRNA or protein product beneficial to the host. The benefit may be, forexample, to improve health, protect against infection, or remedy adeficiency. The marker may enable one to track the location, the levelof replication, the level of propagation, the level of transcription, orthe level of translation of the virus or its products or components.Suitable markers include those which are readily detectable, such asfluorescent proteins, chromogenic proteins, etc. Optionally, a secondagent may be used or added for detection of the marker protein or fordevelopment of a detectable substance. Introduced genes may be human ornon-human, heterologous (from another species) or homologous (from samespecies) or endogenous (from the same subject).

Any DNA viral genome can be used, whether single stranded or doublestranded. Examples of suitable viruses which may be used, includewithout limitation, wild-type or variants of adeno-associated virus(AAV), adenovirus, herpes simplex virus, varicella, variola virus,hepatitis B, cytomegalovirus, JC polyomavirus, BK polyomavirus,monkeypox virus, Herpes Zoster, Epstein-Barr virus, human herpes virus7, Kaposi's sarcoma-associated herpesvirus, human parvovirus B19, andenterovirus. The virus may be, without limitation, cytotoxic, cytolytic,or cause latent infections. Viral vectors in which viral genomes havebeen modified may also be used. As an example, a genome that is modifiedto encode fewer viral proteins may be used. As a further example, aviral genome that is modified to encode no viral proteins may be used.Viral genomes may include, by way of non-limiting example, invertedterminal repeats and/or other non-coding genetic elements thatfacilitate packaging of engineered viral genomes into the capsid.

Viral genomes may be delivered to a mammalian host cell as naked DNA, ina liposome, complexed to a polymer, in a condensed or compacted state,in a gold particle, in a virion, or any other means that is suitable forthe application. Typically a complete viral genome will be administered,but in some situations, it may be desirable to use a partial genome. Thepartial genome may be complemented by helper functions provided by thehost cell or another genomic or viral entity. Partial genomes may beused, for example, if the therapeutic payload is large and someessential viral functions must be omitted to package.

Recombinant viruses may be administered to a mammal or mammalian cellsaccording to any route which is effective for the purpose. Theadministration may be systemic, e.g., via the blood. It may be deliveredorally, subcutaneously, topically, bucally, anally, intramuscularly,intravenously, intratumorally, intracranially, intrathecally,subretinally, etc. Any suitable carrier or vehicle may also be used foradministration. It may be desirable to pre-treat the cells or mammal torender them more permeable to or receptive to the recombinant virus. Amammal “in need of” a recombinant virus may be one for whom the viruswill be beneficial. It may be a mammal with a disease or deficiency. Itmay be one for whom a diagnosis or analysis will be made. It may be onewho can benefit from the administered recombinant virus, even though itdoes not have a disease or deficiency.

Taken together, our results show that incorporation of c41 or humantelomeric sequences into the AAV genome (a) does not lower viralpackaging and infectivity, (b) prevents TLR9-mediated inflammation, (c)reduces induction of pro-inflammatory cytokines, and (d) increasestransgene expression. The increased transgene expression may be due to areduced immune response, as TLR9 activation also induces interferonexpression, which triggers an antiviral state. The engineeredimmune-evasion property we show below is specific (against TLR9),transient (e.g., may occur during viral entry), and does not result insystemic immune suppression (only targets AAV-infected immune cells).

The inhibitory nucleic acid sequences we used, as well as others knownin the art, may be incorporated into other viruses that, like AAV, havepotential utility for humans and other mammals but elicitinflammatory/immune responses that may be undesirable. For example,oncolytic viruses that preferentially infect and lyse cancer cells areused to kill or shrink tumors. These viruses are replicative (unlike AAVvectors used for gene therapy) so they can release new virions to shrinkthe remaining tumor. Examples include wild-type or variants of herpessimplex virus, adenovirus, and enterovirus. Some reports have shown thatimmunosuppression by chemotherapy can enhance oncolytic virus therapy,as the immune system normally attempts to inactivate the oncolyticvirus, which would prevent it from infecting cancer cells. Therefore, itis possible that incorporating inhibitory oligonucleotides in thegenomes of oncolytic viruses like herpes simplex virus may allow it toevade immune clearance and persist longer for oncolysis.

The above disclosure generally describes the present invention. Allreferences disclosed herein are expressly incorporated by reference. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention. The disclosure of the invention includes all embodimentsexplicitly recited in the claims. Additionally, all features disclosedin the dependent claims apply equally to the independent claim fromwhich they are based as to the other independent claims. Thus suchcombinations of dependent claims with other independent claims areexpressly contemplated and disclosed.

Example 1—Construction of Modified Viral Genome

To engineer an AAV vector that has the ability to specifically evadeTLR9 activation in immune cells, we inserted two copies of c41 separatedby a 5-nucleotide-long spacer (AAAAA; SEQ ID NO: 8) into the 3′untranslated region of AAV vector encoding enhanced green fluorescentprotein (eGFP) (FIG. 3B). Subsequently, we produced wild-type AAV-eGFPvirus and AAV-eGFP-c41 virus (harboring two c41 insertions). Infectioustiters of both viruses were comparable (˜10⁹ infectious units/ml, asdetermined by titering on HeLa cells), suggesting that addition of c41into the viral genome did not hamper viral packaging and infectivity, animportant consideration for viral vectors that have to be mass-producedfor gene therapy.

Example 2—Modified Viral Genome Reduces NF-kB Activation

To measure the inflammatory response, we used HEK293 cells stablyexpressing TLR9 (HEK293 TLR9 cells), which senses AAV DNA genomes, andalso expressing alkaline phosphatase under the transcriptional controlof NF-kB. When NF-kB is activated, which indicates inflammation,alkaline phosphatase is secreted into the media and acts on a providedsubstrate, leading to a change in color of the media that can bemeasured on a plate reader. We mock-infected HEK293 TLR9 cells orinfected them with either AAV-eGFP or AAV-eGFP-c41. In agreement withthe literature, AAV-eGFP infection induced a small but statisticallysignificant increase in NF-kB activity (FIG. 4). In contrast,AAV-eGFP-c41 infection was not significantly different compared tomock-infected cells, indicating that the virus was able to evadeeliciting an inflammatory response.

Example 3—Modified Viral Genome Transduced More Cells and Expresses MoreTransgene

We analyzed the above three conditions (described in FIG. 4) for eGFPexpression using flow cytometry. We found that AAV-eGFP-c41 transducedmore cells than AAV-eGFP (52.7% GFP+ compared to 34.6% GFP+) (FIG. 5).In addition, GFP+ cells from AAV-eGFP-c41 infection expressed ˜twice asmuch eGFP as GFP+ cells from AAV-eGFP infection (mean fluorescenceintensity [MFI] of 5335 compared to 2749).

In summary, we engineered an AAV vector to evade TLR9-mediatedinflammation by incorporating an inhibitory oligonucleotide in the viralgenome.

Example 4—Incorporation of c41 or Telomeric Sequences in AAV GenomeTransduced More Cells and Reduced TNF Induction

We inserted three copies of “telomere,” a sequence derived frommammalian telomeres that contains the suppressive (TTAGGG)₄ motif (SEQID NO: 6), which has been shown to block TLR9 signaling (FIG. 6A andFIG. 6B). AAV-eGFP-telomere virus yielded similar viral titers asAAV-eGFP and AAV-eGFP-c41 when titered on HeLa cells, demonstrating thatincorporation of “telomere” does not hinder viral packaging andinfectivity.

When we infected a B cell line with similar amounts of AAV-eGFP orAAV-eGFP-c41 or AAV-eGFP-telomere virus, both AAV-eGFP-c41 andAAV-eGFP-telomere viruses transduced more cells than AAV-eGFP (FIG. 7A).This finding suggests too that incorporation of inhibitory sequences inthe genome of AAV increases transgene expression.

Subsequently, we harvested primary human CD14+ monocytes from blood andsubjected them to similar infection conditions as above. We performedELISA on the supernatant to analyze TNF production, as TNF is aprototypical pro-inflammatory cytokine induced by NF-kB activation.AAV-eGFP infection increased TNF production compared to mock-infection,while AAV-eGFP-c41 and AAV-eGFP-telomere infections showed no or littleincrease in TNF production (FIGS. 7A-7B), showing that the two viruseswere able to evade eliciting inflammatory responses.

Example 5—Engineering a Self-Complementary AAV Vector

Investigators often use short inhibitory oligonucleotides (typically10-30 nucleotides in length) to antagonize TLR9 signaling in cellculture. However, it is unknown if these inhibitory oligonucleotidesretain functionality in the context of a much larger viral genome (i.e.,the sequence is covalently linked on both ends to much longersequences). To test this possibility, we utilized a self-complementary(sc) AAV vector encoding enhanced green fluorescent protein (eGFP), andinserted 3 copies of “c41” or “telomere”, derived from bacteria andmammalian telomeres respectively [52, 57, 58, 61], into a plasmidharboring the vector genome (FIGS. 8A and 8B). We started with sc AAVvectors as they have been shown to be more efficient at triggering TLR9activation and inducing more inflammation in the mouse liver thansingle-stranded (ss) AAV vectors. As “c41” and “telomere” are predictedto have strong secondary structure, we used an AAAAA linker betweencopies of the inhibitory oligonucleotide. In addition, 3×c41 and 3×telomere sequences were placed after the polyA sequence and upstream ofthe right inverted terminal repeat (ITR) so they would be present in theDNA genome during viral entry, but would be absent from subsequent mRNAtranscripts upon successful transduction (“scAAV-eGFP-3×c41” and“scAAV-eGFP-3× telomere”). Finally, to determine if the location ofinhibitory oligonucleotide in the viral genome matters, we also createda vector where 3× telomere was located between the left ITR and thepromoter (“scAAV-3× telomere-eGFP”).

Example 6—Inflammatory Responses in Primary Human Macrophages andMonocytes In Vitro

We packaged the various AAV vectors into AAV2 serotype and infectedprimary human monocyte-derived macrophages at a multiplicity ofinfection (MOI) of 10⁵ viral genomes (vg) per cell. As expected, wefound that scAAV-eGFP infection of macrophages elicited robust inductionof TNF in the supernatant, a prototypical inflammatory cytokine withwell-described roles in stimulating fever, apoptosis and inflammation,and is produced upon TLR9 signaling and NF-kB activation (FIG. 9A). Incontrast, both scAAV-eGFP-3×41 and scAAV-eGFP-3× telomere markedlydecreased TNF induction by >95%, indicating that incorporation of “c41”or “telomere” in these viruses could evade eliciting inflammatoryresponses compared to the wild-type (WT) vector. Furthermore, scAAV-3×telomere-eGFP was also able to prevent TNF induction by >95%,demonstrating that the inserted inhibitory oligonucleotides can beplaced in other parts of the viral genome and retain the ability toblock inflammation. Mock infection with phosphate-buffered saline (PBS)and treatment with ODN 2006, a commercially available CpG-containingoligonucleotide that is known to strongly activate TLR9/NF-kB andinflammation, served as negative and positive controls respectively. Wetested primary human CD14+ monocytes and found that again, scAAV-eGFPtriggered robust TNF induction while scAAV-eGFP-3×c41 and scAAV-eGFP-3×telomere negated most of the TNF induction (FIG. 9B). scAAV-3×telomere-eGFP likewise reduced TNF induction, although inhibition was˜85%, which may be due to differences between cell types or donortissue. The evasion of TNF induction was also reproduced in primaryCD14+ monocytes obtained from another donor (FIG. 9C).

As further characterization, we inserted 3 copies of “control,” a randomsequence that does not block TLR9, or 1 copy of “telomere”, into aplasmid harboring the vector genome (FIG. 10A, FIG. 10B). We picked thesequence “control” as it has been used as a negative controloligonucleotide in TLR9 experiments. We found that scAAV-eGFP-lxtelomere was able to reduce TNF induction compared to scAAV-eGFP inhuman macrophages, but not as efficiently as scAAV-eGFP-3× telomere(FIG. 10C). This indicates that 1 copy of telomere can reduceinflammation. We also observed that scAAV-eGFP-3×control elicited TNFsecretion as efficiently as scAAV-eGFP in human monocytes, suggestingthat insertion of sequences that inhibit TLR9 are required to blockinflammation (FIG. 10D).

As AAV vectors are considered biologics and may exhibit lot-to-lotvariability, we produced another batch of both scAAV-eGFP andscAAV-eGFP-3× telomere AAV2 viruses, and found that scAAV-eGFP-3×telomere was able to reduce ˜75% of TNF induction compared to the WTvector (FIG. 11A). Based on the multiple viral preps and donor monocytesand macrophages (FIGS. 9A-9C, FIGS. 10A-10E and FIGS. 11A-11B), weconclude that our engineered vectors containing 3 copies of “c41” or“telomere” reduce TNF induction by approximately 75-98% compared to theWT vector. On average, scAAV-eGFP-3× telomere reduced ˜85% of TNFinduction compared to scAAV-eGFP. Importantly, we did not observedifferences in viral titers (assayed by qPCR for viral genomes) obtainedfrom producing any of the above AAV2 vectors, suggesting that theengineered vectors are not defective in packaging (data not shown).Furthermore, when we infected HeLa cells, a permissive cell line widelyused to titer AAV infectivity, with a range of MOIs of scAAV-eGFP andscAAV-eGFP-3× telomere, we did not observe differences in transduction(% GFP+ cells) over 4 logs of viral titers, demonstrating that theengineered vector is equally competent at transducing cells (FIG. 11B).

Example 7—Inflammatory Responses in Liver Tissues of Mice In Vivo

Intravenous delivery of AAV is often used to transduce hepatocytes forgene therapy. Previous work has shown that upon intravenousadministration of AAV, Kupffer cells (resident hepaticantigen-presenting cells) in the liver of mice are capable of sensing scAAV genomes and triggering inflammatory and innate immune responses 1-9h later [36]. These responses include induction of proinflammatorycytokines such as TNF and IL6 and type I interferons such as IFN-β.TLR9−/− mice do not exhibit these inflammatory and innate immuneresponses in the liver, demonstrating a central role for TLR9 in vivo asan innate immune sensor. In addition, immune cells such as neutrophils,macrophages and natural killer (NK) cells infiltrate the liver 2 h afterAAV administration. To determine if our engineered vectors can reduceinflammation in the liver in vivo, we administered PBS or equal amountsof scAAV-eGFP or scAAV-eGFP-3× telomere via tail vein injection. Weselected scAAV-eGFP-3× telomere for in vivo characterization as“telomere” is derived from human sequences and might be preferable forclinical use. In agreement with previous work, scAAV-eGFP stimulatedincreased Tnf and 116 expression in the liver (approximately 3 to 10fold, compared to saline), indicating inflammation (FIG. 12A). Incontrast, scAAV-eGFP-3× telomere showed little to no increase ininflammatory markers. We tested more mice in subsequent experiments andfound that scAAV-eGFP stimulated statistically significant Tnf inductionin the liver compared to saline, while scAAV-eGFP-3× telomere andscAAV-eGFP-3×c41 did not (FIGS. 12B and 12C), demonstrating theirability to evade eliciting inflammation in the liver. Finally, weconfirmed that scAAV-eGFP-3×control is not able to prevent inflammationin the liver compared to scAAV-eGFP (FIG. 10E).

Example 8—Engineering a Single-Stranded AAV Vector and DeterminingInflammatory Responses in Eye Tissues of Mice In Vivo

Next, we engineered a single-stranded AAV vector, ssAAV-eGFP, byinserting 5 copies of “telomere” with AAAAA linkers, followed by another5 copies but in anti-sense orientation, into the plasmid, givingssAAV-eGFP-5× telomere (FIG. 13). Since both positive and negativestrands of the viral genome are equally likely to be packaged into aviral particle, this ensures that each packaged viral genome would have5 copies of “telomere” in the correct orientation. Two AAV8 viruses wereproduced and purified. Again, we did not observe differences in titersbetween the two vectors, suggesting similar packaging efficiency (datanot shown). ssAAV-eGFP was selected as it has been previously used forsubretinal injections in mice and efficiently transduces photoreceptorsin the eye [62].

Several studies have suggested that AAV gene therapy in the eye andbrain appears to be generally safe [63]. While the eye is often assumedto be an immune-privileged site, it is known to harbor microglia,resident macrophages of the central nervous system which have beenreported to express TLR9 and respond to CpG motifs [64-67]. A recentstudy delivering AAV vectors by subretinal injection in cynomolgusmacaques reported dose-related anterior and posterior segmentinflammation in the animals, and a macaque was euthanized prematurelydue to severe ocular inflammation [68]. Furthermore, vitreous aspiratefrom the euthanized animal demonstrated the presence of neutrophils andmacrophages. Another study utilizing canine models similarly observedanterior and posterior uvetitis upon subretinal injection of AAVvectors, and 3 of 17 eyes developed a multifocal chorioretinitis, whichwas likewise associated with higher vector doses [69]. These findingsstrongly suggest that AAV vectors are subject to innate immunesurveillance in the eye and can trigger deleterious inflammatory andimmune responses.

Saline or similar amounts of ssAAV-eGFP or ss-AAV-eGFP-5× telomere weredelivered via subretinal injection into neonates eyes and measured theexpression of inflammatory and immune genes. The three mice thatreceived saline injections were uniformly low for Tnf expression in theretina and were set to 1 fold expression (FIG. 14A). In contrast, of thefive mice that received ssAAV-eGFP, two exhibited mild upregulation ofTnf (1.9 fold and 8.3 fold), while three animals demonstrated largeinduction of Tnf (62.2 fold, 534 fold, and 1003 fold), with a mean of321 fold for the five animals. This finding indicates that while thereis variability in inflammation, some animals mount a very stronginflammatory response in the retina upon ssAAV-eGFP subretinalinjection. The variability may be due to differences in each injectionprocedure or the immune status of each animal. Strikingly, the fiveanimals receiving ssAAV-eGFP-5× telomere had a mean Tnf induction of 5.6fold with much less variability, suggesting that ssAAV-eGFP-5× telomerewas able to avoid eliciting strong inflammation. Similar results wereobserved in the rest of the eyecup but at a lower magnitude, indicatinginflammation was not restricted to the retina (FIG. 14B). We alsomeasured Ifng expression in the retina, a type II interferon criticalfor antiviral immune responses [70], and observed a similar pattern(FIG. 14C). Prior studies have suggested that subretinal injection ofAAV may trigger immune cell infiltration in the eye. Therefore, we alsoanalyzed expression of genes that are known to be expressed specificallyin different types of immune cells. We found 18.2 fold higher expressionof Aif1 (encoding Iba1, a specific marker for microglia [71, 72]) inssAAV-eGFP injections compared to saline, suggesting microgliaproliferation and/or activation in the retina (FIG. 15A). In contrast,ssAAV-eGFP-5× telomere only showed 1.9 fold induction of Aif1expression. In addition, we found 45.8 fold and 41.8 fold induction ofCd4 and Cd8a, markers of CD4+ helper T cells and CD8+ cytolytic T cellsrespectively, by ssAAV-eGFP, while ssAAV-eGFP-5× telomere only showed1.5 fold and 3.4 fold induction (FIGS. 15B and 15C). Again, there wasconsiderable variability among mice treated with ssAAV-eGFP with asubset of animals showing robust induction. These results demonstratethat subretinal injection of ssAAV-eGFP administration can stimulate Tcells infiltration in the retina, while ssAAV-eGFP-5× telomere stronglydiminishes it. Taken together, our data indicate that while ssAAV-eGFPinduces robust inflammatory and immune responses in the retina and thesurrounding tissue, and significant variability is observed,ssAAV-eGFP-5× telomere is capable of mitigating a large portion of theseresponses.

Given the marked differences in inflammation both in vitro and in vivo,we sought to determine if there are any differences in long-term geneexpression. We examined flat-mounted eye cups at P30, 29 d aftersubretinal injection of the mice, and found that more cells were GFP+and GFP expression was stronger in ssAAV-eGFP-5× telomere treated eyescompared to ssAAV-eGFP, suggesting enhanced gene expression (FIG. 16).Thus, the engineered vector is able to reduce inflammatory and immuneresponses in the retina and also augment transgene expression.

Example 9—Material and Methods Animals

C57BL/6 mice (male, 7-9 weeks old) were purchased from the JacksonLaboratory and CD1 mice were purchased from Charles River Laboratories.

AAV Vectors

Self-complementary (sc) or single-stranded (ss) AAV vectors were used inthis study. Self-complementary vectors lack the terminal resolutionsequence in one ITR. All vector genomes were flanked by AAV2 ITRs.scAAV-eGFP was purchased from Cell Biolabs (VPK-430) and has beenpreviously described [73]. scAAV-eGFP expressed enhanced greenfluorescent protein (eGFP) from the cytomegalovirus (CMV) promoter, andincluded an SV40 intron and SV40 polyA sequence. ssAAV-eGFP has beenpreviously described [62] and was originally obtained from the HarvardDF/HCC DNA Resource Core (clone ID: EvN000061595). ssAAV-eGFP containeda CMV enhancer/promoter, human β-globin intron, eGFP, and β-globin polyAsequence. The sequences of “c41” (5′-TGGCGCGCACCCACGGCCTG-3′; SEQ IDNO: 1) derived from Pseudomonas aeruginosa and “telomere”(5′-TTTAGGGTTAGGGTTAGGGTTAGGG-3′; SEQ ID NO: 9; initial T nucleotide isoptional for function) derived from mammalian telomeres have beendescribed [52, 57, 58, 61]. A widely used “telomere” oligonucleotide(manufactured by Invivogen, catalog code “tlrl-ttag”) harbored anadditional T (in bold) compared to published studies and thus wasincluded in the sequence. During the course of this study, Invivogenremoved the additional T in their manufactured “telomere”oligonucleotide (catalog code “tlrl-ttag151”). In addition, “control”(5′-GCTAGATGTTAGCGT-3′; SEQ ID NO: 34) was used as a negative controlsequence that does not inhibit TLR9 activation (Invivogen, catalog code“tlrl-2088c”).

To engineer scAAV-eGFP, sequences were inserted into the unique SpeIsite found immediately 5′ of the right ITR. To facilitate sub-cloning, aunique ClaI site was created immediately 5′ of the inserted sequences,thus allowing ClaI/SpeI sub-cloning of sequences. 3 copies of “c41,”“telomere,” or “control” were inserted, separated by AAAAA linkers,giving scAAV-eGFP-3×c41, scAAV-eGFP-3× telomere and scAAV-eGFP-3×control, respectively. Alternatively, one copy of “telomere” wasinserted, with an AAAAA linker (SEQ ID NO: 8), giving scAAV-eGFP-lxtelomere. We also inserted 3× telomere between the left ITR and CMVpromoter using the unique AvrII site, giving scAAV-3× telomere-eGFP.

To engineer ssAAV-eGFP, KpnI-5× telomere(sense)-5×telomere(anti-sense)-NheI was inserted immediately 5′ of the XbaI siteadjacent to the right ITR. Again, AAAAA was used as a linker betweencopies of “telomere”. Both sense and anti-sense sequences of “telomere”were added as single-stranded AAV vectors have an equal chance ofpackaging positive or negative strands of the viral genome, thusensuring that all packaged AAV genomes will carry 5 copies of “telomere”in the right orientation.

Self-complementary vectors were packaged into AAV2 (Vigene Biosciences)by triple transfection of HEK293 cells and purified using iodixanolgradient ultracentrifugation and then concentrated to 500 ul usingAmicon Ultra-15 columns in PBS. The purified viruses were titered byqPCR using primers derived from ITR and an AAV standard. The final yieldof the viruses ranged from 0.5-3×10¹³ vg.

Single-stranded vectors were packaged into AAV8 based on previouslydescribed protocols [74, 75]. Briefly, AAV vector, rep2-cap8 packagingplasmid and adenoviral helper plasmid were transfected into HEK293Tcells with polyethylenimine and supernatant was collected 72 h aftertransfection. AAV8 viruses were precipitated with 8.5% w/v PEG8000 and0.4M NaCl and centrifuged at 7000 g. The pellet was resuspended in lysisbuffer (150 mM NaCl and 20 mM Tris, pH 8.0) and MgCl2 was added to afinal concentration of 1 mM. The resuspended viruses were incubated with25 U/ml Benzonase (Sigma) at 37° C. for 15 min and run on an iodixanolgradient. Recovered AAV vectors were washed 3 times with PBS usingAmicon 100K columns (EMD Millipore) and concentrated to 100-500 ul ofPBS. Protein gels were run to determine virus titers, using serialdilutions of previous AAV standards for comparison.

Primary Human Monocytes and Monocyte-Derived Macrophages for In VitroStudies

Human peripheral blood mononuclear cells (PBMCs) from unidentifiedhealthy donors were purchased (ZenBio). This study was done inaccordance with the ethical guidelines of Harvard Medical School. CD14+monocytes were positively selected from PBMCs using anti-CD14 magneticmicrobeads according to the manufacturer's instructions (MiltenyiBiotec) or purchased from Stemcell Technologies. To obtainmonocyte-derived macrophages, monocytes were cultured with 50 ng/ml ofrecombinant human macrophage colony stimulation factor (rhM-CSF,purchased from Peprotech) for 5 to 6 d to allow differentiation intomacrophages. Monocytes and macrophages were either used fresh orcryopreserved for subsequent studies.

1×10⁵ monocytes or macrophages were seeded in 190 ul of RPMI growthmedia per well in 96 well round bottom plates or 96 well flat bottomplates respectively, and infected with 10 ul AAV2 viruses at indicatedMOIs in PBS. Mock infection (addition of 10 ul PBS) and ODN 2006 (finalconcentration of 5 uM, Invivogen), a CpG-containing oligonucleotideknown to activate TLR9 and trigger inflammation, served as negative andpositive controls. 18 h after infection, supernatants were collected andclarified by low speed centrifugation, followed by ELISA for human TNF(Thermo Scientific).

HeLa Cells Infection

HeLa cells are highly permissive for AAV2 vectors and are commonly usedto determine the transducing titer of AAV2 vector preparations [76].Briefly, HeLa cells were seeded overnight in 12 wells and wereapproximately 80% confluent at time of infection (3×10⁵ cells). Cellswere infected with serial ten-fold dilutions of viruses at indicatedMOIs and incubated for 48 h before fixing with 1% paraformaldehyde inPBS and followed by flow cytometry analysis for GFP+ cells. PBSmock-infected cells were used to determine GFP+ signal.

Liver Studies In Vivo

Adult C57BL/6 mice were injected intravenously with 100 ul PBS or AAV2viruses (10¹¹ vg per animal) by tail vein injection as previouslydescribed [36]. 2 h later, the animals were sacrificed and a portion ofthe right median lobe of the liver was saved in RNAlater solution(Thermo Scientific). Total RNA was extracted from 10-30 mg ofmechanically disrupted liver sample by using an RNA extraction kit(OMEGA Bio-Tek). Similar amounts of RNA were reverse transcribed intocDNA with a high-capacity RNA-to-cDNA kit (Thermo Scientific) andsimilar amounts of cDNA were assayed with quantitative PCR (qPCR) usingTaqMan Fast Advanced Master Mix (Thermo Scientific) and commerciallyavailable pre-designed primers/probes with FAM reporter dye for theindicated target genes (IDT). Expression level for each gene wascalculated by normalizing against the housekeeping genes Actb or Gapdhusing the AACT method and expressed as fold levels compared tosaline-injected mice. All qPCR reactions were run on a realplex⁴Mastercycle (Eppendorf).

Eye Studies In Vivo

Subretinal injection into postnatal day 1 (P1) CD1 neonate eyes wereperformed as previously described [74, 75]. Approximately 0.2 ul AAV8virus (1.8×10⁸ vg per eye) was introduced into the subretinal spaceusing a pulled angled glass pipette controlled by a FemtoJet(Eppendorf). At P21, animals were sacrificed and the eyecup wasdissected out. The retina and the rest of the eyecup were subjected toRNA extraction, reverse transcription, and qPCR as described in theliver studies. To visualize GFP expression by histology, eyes wereexcised at P30, fixed in 4% paraformaldehyde for 2 h, and washed in PBS3 times. Eye cups were dissected out by removing the cornea, lens, iris,vitreous body and peripheral muscles. Images of flat-mounted eye cupswere taken using a ×10 objective on a Keyence BZ-x700 microscope. Imagesused for comparison between groups were taken at the same imagingsettings in the same imaging session.

Statistics

Unpaired two-tailed Student's t-tests were used to compare differencesbetween two unpaired experimental groups in all cases. A P value of<0.05 was considered statistically significant. No pre-specified effectsize was assumed and in general three to five replicates for eachcondition was used.

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1. (canceled)
 2. A recombinant viral genome comprising four repeatedmonomers of TTAGGG (SEQ ID NO: 6).
 3. The recombinant viral genome ofclaim 2, wherein the recombinant viral genome is an adeno-associatedvirus (AAV) genome.
 4. The recombinant viral genome of claim 2, whereinthe recombinant viral genome is selected from the group consisting ofadenovirus, herpes simplex virus, varicella, variola virus, hepatitis B,cytomegalovirus, JC polyomavirus, BK polyomavirus, monkeypox virus,Herpes Zoster, Epstein-Barr virus, human herpes virus 7, Kaposi'ssarcoma-associated herpesvirus, and human parvovirus B19.
 5. Therecombinant viral genome of claim 2, wherein the recombinant viralgenome is single stranded.
 6. The recombinant viral genome of claim 2,wherein the viral genome is packaged in a virion.
 7. The recombinantviral genome of claim 2, wherein the recombinant viral genome comprisesa therapeutic gene.
 8. The recombinant viral genome of claim 2, whereinthe recombinant viral genome is a cytotoxic virus. 9-14. (canceled) 15.The recombinant viral genome of claim 2, wherein the recombinant viralgenome comprises a non-human gene.
 16. The recombinant viral genome ofclaim 2, wherein the four repeated monomers of TTAGGG (SEQ ID NO: 6) areinserted downstream of or in a 3′ untranslated region of the viralgenome.
 17. The recombinant viral genome of claim 2, wherein therecombinant viral genome is covalently linked by a phosphodiester bondto a nucleic acid sequence comprising the four repeated monomers ofTTAGGG (SEQ ID NO: 6).
 18. The recombinant viral genome of claim 2further comprising a detectable marker.
 19. The recombinant viral genomeof claim 18, wherein expression of the detectable marker is inducible.20. The recombinant viral genome of claim 2 comprising the nucleotidesequence of SEQ ID NO:
 9. 21-42. (canceled)
 43. The recombinant viralgenome of claim 2 comprising three repeated monomers of the nucleotidesequence of SEQ ID NO:
 9. 44. The recombinant viral genome of claim 2comprising five repeated monomers of the nucleotide sequence of SEQ IDNO:
 9. 45. A method of treating a mammal, comprising: administering tothe mammal the recombinant viral genome of claim
 2. 46-73. (canceled)74. A method of producing a recombinant viral genome, comprising:inserting into a viral genome four repeated monomers of TTAGGG (SEQ IDNO: 6) or the nucleotide sequence of SEQ ID NO:
 9. 75. A nucleic acidvector comprising the nucleotide sequence of SEQ ID NO:
 9. 76-90.(canceled)
 91. The recombinant viral genome of claim 2, wherein therecombinant viral genome is self-complementary.
 92. A recombinant viralgenome comprising a sequence that is at least 95% identical to thesequence of SEQ ID NO: 9.